Galectin-3 is a protein (Mr 30,000), initially identified and purified on the basis of its galactose-specific carbohydrate-binding activity, recently documented to be a required factor in nuclear pre-mRNA splicing. The key observations made include: a) nuclear extracts derived from HeLa cells, capable of carrying out splicing in a cell-free assay, contain galectin-3; b) nuclear extracts depleted of galectin-3 by affinity adsorption on lactose-agarose become deficient in splicing; c) the activity of the lactose-agarose-depleted extract could be reconstituted by addition of purified recombinant galectin-3; and d) saccharides that bind galectin-3 with high affinity inhibited the splicing reaction. Inside cells, galectin-3 is found in both the cytoplasm and the nucleus. There are two isoforms of the protein: a) a pI 8.7 unmodified polypeptide; and b) a pI 8.2 form, representing the polypeptide modified by a single phosphate. The phosphorylated (pI 8.2) form is found in both the cytosol and the nucleus, whereas the unmodified polypeptide (pI 8.7) is found exclusively in the nucleus. Recently, we have developed a permeabilized cell assay to monitor the export of galectin-3 from the nucleus. While the nuclear residue contained both the pI 8.2 and pI 8.7 forms, only the phosphorylated (pI 8.2) polypeptide was being exported. These results suggest a requirement for the phosphorylation of the galectin-3 polypeptide prior to its export from the nucleus. On the basis of these results, the objectives of our research include: a) to establish the site(s) of phosphorylation of the murine galectin-3 polypeptide; b) to carry out site-directed mutagenesis to derive mutant polypeptides that cannot be phosphorylated (e.g., Ser to Ala; Ser to Asp, etc.); and c) to compare the nuclear versus cytoplasmic distribution of galectin-3 in cells transfected with the wild-type and mutant constructs. Mass spectrometric methods will be used to identify the site of phosphorylation in this study.